In order to explore functional properties of the human erythrocyte membrane in health and disease, we plan to do the following experiments. (1) Study endocytosis in intact RBC and ghosts. Specifically we will study: the distribution of actin in the cytoskeleton of white ghosts as endocytosis proceeds using monoclonal anti-actin antibody; the role of transglutaminases in RBC endocytosis using the transglutaminase inhibitors histamine, dansylcadaverine, and bacitracin; the protein composition of the endocytic vacuoles produced in intact RBC. (2) Calcium in RBC appears to exist both in cytosol and membrane associated pools and endocytosis inducing agents cause a shift of calcium from cytosol to membrane associated sites. We propose to identify the location of the membrane associated calcium because an interaction of calcium with specific membrane constituents could result in important modifications of membrane functions. (3) The presence of an actin-activated ATPase in the RBC membrane raises the possibility that it is a myosin-like molecule. Final proof of this identity requires that we solubilize and partially purify this activity and show that it reacts with anti-myosin antibodies that we have raised to purified human uterine myosin. (4) RBC can now be stored for transfusion for 35 days. Future prolongation of the storage period may depend on the avoidance of a putative membrane storage lesion. Our current experiments involve the storage of human RBC, followed by determination of in vivo RBC survival, and correlation of this finding with in vitro measurements of drug induced endocytosis and analysis of acrylamide gel electrophoretic patterns of RBC membrane proteins. (5) There are hemolytic disorders due to membrane abnormalities. In hereditary pyropoikilocytosis (HPP) drug induced endocytosis is remarkably enhanced. Spectrin may be unstably anchored in HPP membrane and we propose to test this hypothesis by measuring the redistribution of spectrin in the HPP membrane during white ghost endocytosis using polyclonal rabbit anti-spectrin in a ferritin sandwich.